Transfer RNA-mediated antitermination <em>in vitro</em>

Harald Putzer

Ciarán Condon

Dominique Brechemier-Baey

Renata Brito

Marianne Grunberg-Manago

CNRS – UPR 9073, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France

^{To whom correspondence should be addressed. Tel: +33 1 58415123; Fax: +33 1 58415020; Email:}rf.cpbi@reztup

Received 2002 Apr 26; Revised 2002 May 16; Accepted 2002 May 16.

Abstract

The threonyl-tRNA synthetase gene (thrS) is a member of the T-box family of ∼250 genes, found essentially in Gram-positive bacteria, regulated by a tRNA-dependent antitermination mechanism in response to starvation for the cognate amino acid. While interaction between uncharged tRNA and the untranslated leader region of these genes has been firmly established by genetic means, attempts to show this interaction or to reconstitute the antitermination mechanism in vitro using purified tRNAs have so far failed. In addition, a number of conserved sequences have been identified in the T-box leaders, for which no function has yet been assigned. This suggests that factors other than the tRNA are important for this type of control. Here we demonstrate tRNA-mediated antitermination for the first time in vitro, using the regulatory tRNA^{isoacceptor isolated from}Bacillus subtilis and a partially purified protein fraction. As predicted by the model, aminoacylation of tRNA^{with threonine completely abolishes its ability to act as an effector. The role of the partially purified protein fraction can be functionally substituted by high concentrations of spermidine. However, this polyamine does not play a significant role in the induction of}thrS expression in vivo, suggesting that it is specific protein co-factors that promote T-box gene regulation in conjunction with uncharged tRNA.

Abstract

ACKNOWLEDGEMENTS

We are grateful to O. Pellegrini for the purification of RNA polymerase, to A. Sekowska and A. Danchin for the strain BSIP7010 and to D. Bechhofer for the chromosomal DNA of strain BD2411. We thank M. Springer and P. Stragier for helpful discussions. R.B. was supported by the ERASMUS program. This work was supported by funds from the CNRS (UPR 9073), MRE (Contract 92C0315), Université Paris VII (Contract DRED) and PRFMMIP from the Ministère de l’Education Nationale.

ACKNOWLEDGEMENTS

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